PROJECT SUMMARY Yersinia pestis, the etiological agent of plague, is mainly transmitted to humans by fleabite. Replication and biofilm formation by Y. pestis in the flea gut are essential steps for flea foregut blockage, a condition which facilitates subsequent transmission of Y. pestis to a mammalian host. Flea-borne transmission thus requires that Y. pestis rapidly adapt to, and overcome, physiological and immunological barriers imposed by two distinctive host environments ? first the flea and subsequently the mammal. How flea gut adaptation is regulated to produce a transmissible infection and consequent transmission efficiency represents a significant gap in our knowledge, and understanding this regulation can provide new opportunities to block flea-borne transmission of plague. Toward understanding the regulatory mechanisms necessary for flea-borne transmission of Y. pestis, we have produced evidence that supports testing the hypothesis that Y. pestis biofilm formation in the flea gut is regulated post-transcriptionally by small non-coding RNAs (sRNA). Besides showing that the sRNA chaperone protein, Hfq, is required for biofilm blockage formation and optimal survival and fitness of Y. pestis in the flea gut, we have: (1) identified a unique flea-specific intergenically coded repertoire of sRNAs that are produced by Y. pestis during flea infection; and (2) showed that a Y. pestis mutant of a known sRNA is deficient in its ability to form biofilm blockage in fleas and therefore to transmit the plague agent. Post-transcriptional regulation of sRNAs is facilitated by complementary base pairing to a target mRNA, thereby altering mRNA target stability or translation. Hence, the goals of this study are to: (Aim 1) determine if flea-specific sRNAs regulate the unique Y. pestis biofilm and adaptation to the flea gut; (Aim 2) determine if flea-specific sRNAs prime successful flea-borne transmission of Y. pestis to its mammalian host; and (Aim 3) identify the cognate mRNA targets of sRNAs and characterize the functional interaction between sRNAs and their cognate mRNAs. Cumulatively this data will result in identification of novel transmission and virulence mechanisms in plague transmission that may presumably be conserved in other vector-borne bacterial pathogens.